Vitamin K-dependent (VKD) carboxylation, an essential post-translational modification catalyzed by gamma-glutamyl carboxylase (GGCX), is required for the biological functioning of proteins that control blood coagulation, vascular calcification, bone metabolism, and other important physiological processes. Concomitant with carboxylation, reduced vitamin K (KH2) is oxidized to vitamin K epoxide (KO). Since humans cannot synthesize vitamin K, KO must be converted back into KH2 in a two-step reduction to complete the vitamin K cycle; this reduction is accomplished by the enzyme vitamin K epoxide reductase (VKOR) and, as we hypothesize, vitamin K reductase (VKR). Despite significant progress in understanding of the enzymes in the vitamin K cycle, fundamental questions remain: 1) Why do some mutations of GGCX result in the bleeding disorder referred to as combined vitamin K-dependent coagulation factors deficiency (VKCFD), while others are linked with Pseudoxanthoma elasticum (PXE)-like syndrome? 2) What are the identities of the VKR enzymes? 3) What is the mechanism for VKOR active site regeneration? 4) Does VKORC1L1, the paralogous enzyme of VKOR, contribute to the vitamin K cycle under physiological conditions? The current proposal aims to identify and characterize the unknown components of the vitamin K cycle, to understand how these various components contribute to VKD carboxylation in the cellular milieu, and to determine how naturally-occurring GGCX mutations contribute to different disease states. To accomplish these goals, we propose the following specific aims. Aim 1: To study GGCX function using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 mediated GGCX knockout cells, in order to understand how GGCX mutations are related to VKCFD and PXE-like syndromes. Aim 2: To characterize and identify VKR using genome-scale CRISPR-Cas9 knockout loss-of-function screening in our HEK293 reporter cell line. Aim 3: To characterize VKOR, naturally-occurring VKOR mutants, and VKORC1L1 in our double-gene knockout HEK293 reporter cells. Information derived from these studies will help us understand how the various vitamin K cycle components contribute to these complex mechanisms, thereby gaining new therapeutic insights into the control of thrombosis and improving therapies for vitamin K-related disorders.